In the ovary, transport of cholesterol to the mitochondrial cytochrome P450 side-chain cleavage enzyme (P450scc) is thought to be the rate- limiting step in steroid production. Since cholesterol is insoluble in the cell, it must be transported to the P450scc via a carrier molecule which facilitates cholesterol's movement across the mitochondrial membrane for steroid production. While the steroidogenic acute regulatory (StAR) protein is theorized to mediate cholesterol transport to the inner mitochondrial membrane, the mechanism for StAR-mediated cholesterol movement and the regulatory factors which control StAR mRNA levels in the ovary are uncertain. Gonadotropins have been shown to enhance StAR expression in MA-10 cells and a recent study by our laboratory has demonstrated that hCG stimulation increases StAR mRNA levels 8 to 9-fold in the ovary within 3 hours of treatment (Sandhoff & McLean, 1996a). These results indicate that gonadotropins increase StAR mRNA levels in parallel with an increase in progesterone production. In addition to the positive effects of gonadotropins on ovarian steroid production, altered cholesterol transport to the mitochondria has recently been identified as a key lytic event in the corpus luteum. Studies by Sandhoff & McLean (1996b) suggest that one antisteroidogenic action of PGF2alpha is to decrease StAR mRNA levels, which results in a decline in steroid production. Based on these investigations, we hypothesize that StAR transcription is positively regulated by gonadotropins via steroidogenic factor-1, while PGF2alpha working through PKC acts either indirectly, by reducing cAMP-mediated StAR gene expression, or directly, by reducing StAR expression via the negative regulatory transcription factor, DAX-1 or the Jun/Fos signal transduction pathway. To test this hypothesis, the Aims of this proposal are as follows: Aim I. To determine whether StAR mRNA expression in the ovary is regulated by PKA and PKC signal transduction pathways. Aim II. To determine whether Steroidogenic Factor 1 (SF-1) positively regulates transcription of the rat StAR gene by interaction with multiple putative SF-1 binding sites. Aim III. To determine whether estradiol acting through an estrogen receptor half-site regulates the StAR gene. Aim IV. To determine whether overexpression of DAX-1 or c-Fos negatively regulates StAR transcription. Aim V. To determine whether PKA or PKC activation results in StAR phosphorylation. This study will provide new information concerning ovarian StAR expression and function during luteal development and regression.